More than 80 genes in the human genome code for voltage-gated ion channels and their relatives in the 6TM ion-channel family. Among their many roles these channels mediate pain and other sensory modalities, perform long-distance signaling in the brain, time the heartbeat and control lymphocyte proliferation. They are of great interest as a rich set of potential targets for drugs and therapies, and are also of intrinsic interest as proteins having a uniquely high sensitivity to membrane potential changes. Single-particle electron cryomicroscopy (cryo-EM) is a method for observing the three-dimensional structure of macromolecular complexes. Although the resolution is inferior to X-ray crystallography, cryo-EM technology is making steady progress in this area;meanwhile it has the great advantage of providing "solution structures" of proteins without the necessity of forming crystals. We have developed methods for cryo-EM single-particle imaging of membrane proteins reconstituted into liposomes, and have recently obtained the first closed-state structure of a eukaryotic 6TM channel, the large-conductance Ca2+activated potassium (BK) channel. The structure has relatively low resolution, limited by the small number of particle images we have been able to acquire. In this application we propose first to greatly increase the data-collection efficiency in order to reach resolutions better than 1 nm. We will then image the BK channel in its various conformational states. In parallel, we will apply the methods to Kv1.2, one of the best-studied voltage-gated potassium channels. By creating membrane potentials in the vesicles, we will be able to trap this channel in its closed as well as open states for structur determination. PUBLIC HEALTH RELEVANCE: Voltage-gated ion channels act as molecular switches, controlling the electrical currents in the brain, heart and many other organs. Because there are many (more than 80) varieties, defects in these ion channels give rise to a spectrum of disorders ranging from epilepsy, migraine and muscular paralysis to hypertension and irregular heart rhythm. To understand how they work, we propose to observe the molecular structure of two of these channels, in their various functional states, using novel electron- microscopy techniques.